Polyubiquitination of proliferating cell nuclear antigen by HLTF and SHPRH prevents genomic instability from stalled replication forks

Differential modifications of proliferating cell nuclear antigen (PCNA) determine DNA repair pathways at stalled replication forks. In yeast, PCNA monoubiquitination by the ubiquitin ligase (E3) yRad18 promotes translesion synthesis (TLS), whereas the lysine-63–linked polyubiquitination of PCNA by yRad5 (E3) promotes the error-free mode of bypass. The yRad5-dependent pathway is important to prevent genomic instability during replication, although its exact molecular mechanism is poorly understood. This mechanism has remained totally elusive in mammals because of the lack of apparent RAD5 homologues. We report that a putative tumor suppressor gene, SHPRH, is a human orthologue of yeast RAD5. SHPRH associates with PCNA, RAD18, and the ubiquitin-conjugating enzyme UBC13 (E2) and promotes methyl methanesulfonate (MMS)–induced PCNA polyubiquitination. The reduction of SHPRH by stable short hairpin RNA increases sensitivity to MMS and enhances genomic instability. Therefore, the yRad5/SHPRH-dependent pathway is a conserved and fundamental DNA repair mechanism that protects the genome from genotoxic stress.

Download Full-text

Pivotal roles of PCNA loading and unloading in heterochromatin function

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1721573115 ◽

2018 ◽

Vol 115 (9) ◽

pp. E2030-E2039 ◽

Cited By ~ 12

Author(s):

Ryan Janke ◽

Grant A. King ◽

Martin Kupiec ◽

Jasper Rine

Keyword(s):

Dna Replication ◽

Proliferating Cell Nuclear Antigen ◽

Transcriptional Silencing ◽

S Phase ◽

Nuclear Antigen ◽

Histone Chaperones ◽

Replication Forks ◽

Nucleosome Assembly ◽

Loading And Unloading ◽

Proliferating Cell

In Saccharomyces cerevisiae, heterochromatin structures required for transcriptional silencing of the HML and HMR loci are duplicated in coordination with passing DNA replication forks. Despite major reorganization of chromatin structure, the heterochromatic, transcriptionally silent states of HML and HMR are successfully maintained throughout S-phase. Mutations of specific components of the replisome diminish the capacity to maintain silencing of HML and HMR through replication. Similarly, mutations in histone chaperones involved in replication-coupled nucleosome assembly reduce gene silencing. Bridging these observations, we determined that the proliferating cell nuclear antigen (PCNA) unloading activity of Elg1 was important for coordinating DNA replication forks with the process of replication-coupled nucleosome assembly to maintain silencing of HML and HMR through S-phase. Collectively, these data identified a mechanism by which chromatin reassembly is coordinated with DNA replication to maintain silencing through S-phase.

Download Full-text

PCNASUMO and Srs2: a model SUMO substrate–effector pair

Biochemical Society Transactions ◽

10.1042/bst0351385 ◽

2007 ◽

Vol 35 (6) ◽

pp. 1385-1388 ◽

Cited By ~ 14

Author(s):

H.D. Ulrich

Keyword(s):

Proliferating Cell Nuclear Antigen ◽

Budding Yeast ◽

Effector Proteins ◽

Present Review ◽

Nuclear Antigen ◽

Replication Forks ◽

Replication Factor ◽

Downstream Effector ◽

Active Replication ◽

Proliferating Cell

Attachment of the SUMO (small ubiquitin-related modifier) to the replication factor PCNA (proliferating-cell nuclear antigen) in the budding yeast has been shown to recruit a helicase, Srs2, to active replication forks, which in turn prevents unscheduled recombination events. In the present review, I will discuss how the interaction between SUMOylated PCNA and Srs2 serves as an example for a mechanism by which SUMO modulates the properties of its targets and mediates the activation of downstream effector proteins.

Download Full-text

Continued primer synthesis at stalled replication forks contributes to checkpoint activation

The Journal of Cell Biology ◽

10.1083/jcb.200909105 ◽

2010 ◽

Vol 189 (2) ◽

pp. 233-246 ◽

Cited By ~ 61

Author(s):

Christopher Van ◽

Shan Yan ◽

W. Matthew Michael ◽

Shou Waga ◽

Karlene A. Cimprich

Keyword(s):

Nuclear Antigen ◽

Replication Forks ◽

Ataxia Telangiectasia Mutated ◽

Fixed Amount ◽

Checkpoint Activation ◽

Dna Structures ◽

Egg Extracts ◽

Stalled Replication Forks ◽

Stalled Fork ◽

Proliferating Cell

Stalled replication forks activate and are stabilized by the ATR (ataxia-telangiectasia mutated and Rad3 related)-mediated checkpoint, but ultimately, they must also recover from the arrest. Although primed single-stranded DNA (ssDNA) is sufficient for checkpoint activation, it is still unknown how this signal is generated at a stalled replication fork. Furthermore, it is not clear how recovery and fork restart occur in higher eukaryotes. Using Xenopus laevis egg extracts, we show that DNA replication continues at a stalled fork through the synthesis and elongation of new primers independent of the checkpoint. This synthesis is dependent on the activity of proliferating cell nuclear antigen, Pol-δ, and Pol-ε, and it contributes to the phosphorylation of Chk1. We also used defined DNA structures to show that for a fixed amount of ssDNA, increasing the number of primer–template junctions strongly enhances Chk1 phosphorylation. These results suggest that new primers are synthesized at stalled replication forks by the leading and lagging strand polymerases and that accumulation of these primers may contribute to checkpoint activation.

Download Full-text